Project Summary Genomic imprinting is an epigenetic process resulting in the monoallelic, parent-of-origin- specific expression of a small subset of genes (<150) in the mammalian genome.Imprinted genes are essentialduring and post-embryonic development and defects in dosage imbalance of imprinted genes result in complex rare epigenetic diseases generally involving multiple tissues, named Imprinting Disorders(IDs).Within an imprinted domain, DNA sequence elements named Imprinting Control Regions(ICRs) regulate imprinted expression of genes and are differentially marked by DNA methylation during gametogenesis when maternal and paternal genomes are still in distinct partitions.How ICRs bring about imprinted gene expression and what trans acting protein factors and complexes that occupy parental ICR alleles are required for establishment and maintenance of imprinted patterns of gene expression still remain mostly unknown. The main objective of this proposalis to elucidate the protein assemblies on parental alleles of imprint control regions to understand how cis regulation by trans factors maintain the parent-of- origin expression. We will accomplish this by employing the relatively new methodologies of locus specific genome targeting and intracellular protein labeling. In the first aim, multiple transgenic reciprocal hybrid ES lines will be utilized to decipher the maternal or paternal ICR allele specific protein complexes. In the second aim we will test how depletion of cis acting RNAs and associated loss of ICR enhancer function affects the protein complexes on maternal allele.Finally, in the third aim, we will focus to quantitate the molecular proteomic changes in ES cells with uniparental disomies to understand ICR functions in imprinted disorders. Together, our studies will i) illuminate parental ICR allele specific protein complexes, ii) define ICR derived cis elements required for their protein complex localization/imprinting function and iii) define differential protein complexes in uniparental chromosomal disomies. Findings from this study will lead to a greater understanding of the trans protein factors at Imprint Control Regions and will provide mechanistic details into cis regulation of ICR functions in an unbiased manner.